The overall objective of this project is to define the epigenetic mechanisms of how human Corneal Epithelial (CE) cells respond to growth factors and environmental stresses in the genetic processes of self-renewal and wound healing. We found that EGF/stress-induced activation of the Erk and NF?B signaling pathways subsequently increase/decrease levels of the epigenetic factor CTCF in these cells. Our preliminary data shows that CTCF mediates chromatin remodeling in human CE cells to regulate interactions in the promoter regions between eye-specific Pax6 and 17 identified migration-associated genes, which in turn control CE proliferation, differentiation and wound healing. Thus, larger responses to EGF/stress based on the magnitude of changes in CTCF activities result in altered chromatin remodeling of Pax6 and migration-associated genes that affect CE wound healing. We believe that results of this study will have a significant impact in providing the epigenetic mechanisms underlying CE wound healing and other eye diseases, as we investigate: 1) CTCF- mediated chromatin remodeling to identify the epigenetic effects of EGF/stress stimulation on corneal diseases; 2) Pax6 promoter-linked chromatin interactions to particularly focus on eye specific genes; and 3) migration-associated gene expression and function to understand fundamental mechanisms of CE wound healing. Our central hypothesis is that EGF- and stress-induced chromatin remodeling requires CTCF to assemble Pax6 and associated genes in a group for coordinated expression to determine CE cell migration and proliferation in self-renewal and wound healing. We believe that such novel studies will identify important mechanisms involving epigenetic regulation of gene expression in CE self-renewal and wound healing. We propose three aims: 1) to define how CTCF controls the Pax6 P1 promoter and expression of Pax6 variants in LS/P and CE cells. We propose to determine: what the functional role of the P1 transcripts is; and how CTCF controls P1 promoter activity. We will also determine how Pax6 transcript variants from P0/P1 promoters are differentially expressed in the cornea. 2) To investigate how CTCF mediates chromatin remodeling involving Pax6 and associated genes. We will identify migration-associated genes interacting with Pax6 gene in CTCF-mediated chromatin remodeling and determine functional interactions within the identified genes. 3) To determine EGF/stress-induced CTCF activity alteration on CE migration and wound healing. We will show: how chromatin interacts in the absence of CTCF activity; what changes in expression of the identified genes are induced; and whether wound healing is affected by alterations of CTCF-mediated chromatin remodeling. By achieving the goals of these combined studies, we will provide novel mechanisms that advance understanding of growth factor/stress-induced effects on epigenetic regulation of LS/P and CE cell functions in corneal epithelial self-renewal and wound healing.